Some years ago it was discovered that patients homozygous for a natural mutation (the Δ32 mutation) in the CCR5 gene are generally resistant to HIV infection by blocking virus entry to a cell. Building on this observation, a study published in 2009 reported a potential "cure" in an AIDS patient with leukemia after receiving a bone marrow transplant from a donor with this Δ32 CCR5 mutation. This approach transferred the hematopoietic stem cells (HSC) residing in the bone marrow from the Δ32 donor, and provided a self-renewable and lifelong source of HIV-resistant immune cells. After transplantation, this patient was able to discontinue all anti-HIV drug treatment, the CD4 count increased, and the viral load dropped to undetectable levels, demonstrating an effective transplantation of protection from HIV and suggesting that this approach could have broad clinical utility. But donors with the Δ32 CCR5 mutation are not generally available, and so how could we engineer an analogous CCR5 negative state in human HSC needed for bone marrow transplantation? A potential answer comes from zinc finger nucleases (ZFNs) which have been demonstrated to efficiently block the activity of a gene by cleaving the human genome at a predetermined site and altering the genetic sequence via an error-prone DNA repair process. This modification of the cellular DNA is permanent and can fully block gene function. Recently, ZFNs have been shown to inactivate CCR5 in primary human CD4 T cells, allowing them to preferentially survive and expand in the presence of HIV. A human clinical trial evaluating this approach is on-going, in which patient T cells are re-infused after ZFN-treatment to block CCR5 expression and possibly provide an HIV-resistant reservoir of CD4 T cells. The CIRM Disease Team proposes an approach to modify a patient’s own HSC to circumvent the need to find matched donors that carry the Δ32 CCR5 mutation and yet provide a renewable and long-lasting source of HIV-resistant cells. Testing of this concept is proposed in selected AIDS lymphoma patients who routinely undergo HSC transplantation. Preliminary results in mice transplanted with ZFN-treated HSC show that ZFN-modified, CCR5-negative HSC are functional and support the reconstitution of the immune system. Importantly, after HIV infection, these mice have results similar to those observed in the human patient: (i) reduced viral loads, (ii) maintenance of CD4 T cells in peripheral tissues; and (iii) a powerful selective advantage for the CCR5 negative immune cells. These data support the development of a ZFN approach to treat AIDS patients by first isolating their HSC, modifying them using CCR5-specific ZFNs, and re-infusing them to reconstitute the immune system with CCR5-negative, HIV-resistant immune cells.

Statement of Benefit to California:

California has ~14% of all cases of AIDS in the U.S., and this translates into a medical and fiscal burden larger than any other state except NY. Antiviral chemotherapy accounts for approximately 85% of AIDS-related medical costs, and federal and state law requires that in California the AIDS Drug Assistance Program (ADAP) be the payer of last resort for these medications. In fiscal year 2007-08, the California AIDS Drug Assistance Program (ADAP) served 32,842 clients and filled over 953,000 prescriptions for these clients. The Governor's current spending plan (2009-09 Budget Act) called for $418M to support this program, with funds from several sources including federal (Ryan White Care Act), from an ADAP Rebate Fund, and from the California State General Fund. The ADAP Rebate Fund consists of monies paid to the state by the manufacturers of the drugs provided to the HIV/AIDS clients under the program. The ADAP budget has grown by ~15% yearly for several years, and based on an Legislative Analyst's Office (LAO) review, the problem faced is that, as the case load is increasing, support from the Rebate Fund is decreasing. It is projected by LAO that from a level of $80.3 million at end 2007-08, the Fund will decrease to $24M by 2009-10. The General Fund currently provides $96.3M to the ADAP budget, and it is projected that as the ADAP Rebate Fund shrinks, the shortfall will have to be met by increases from the General Fund by 2011-12. The alternative, as noted by LOA, is to implement cost-cutting measures that would likely increase the barriers to receiving care for some patients, impacting the health of some HIV/AIDS patients and increasing the associated public health risks. The basic problem is that HIV/AIDS is a life-long infection and our current strategy of treatment requires that medication be taken daily for a lifetime. Thus, there is a real need to develop a strategy of treatment that has the potential to reduce the duration of antiviral chemotherapy. This will have significant impact on the quality of life for persons with HIV/AIDS. In this proposal, a cellular therapy derived from genetically modified blood stem cells will be developed and preclinical studies completed, leading to its first evaluation in patients. As important is the benefit that the first test of this technology will have on the overall field of embryonic stem cell research. The ZFN technology used here will have application to other diseases and hurdles surmounted now will benefit future embryonic cell research.

Progress Report:

During the first year of the project, we have made significant progress in meeting the first milestone of the project: Defining the final process of genetically modifying hematopoietic stem/progenitor cells (HSPC) (Item #14, Milestone M3 of Gantt chart). In addition, initial effort has started in Phase II Scale-up/Pre-clinical testing (G15) and more specifically, in hematopoietic stem/progenitor cell processing development (G16).

Some 10 years ago it was discovered that patients homozygous for a natural mutation (the delta 32 mutation) in the CCR5 gene are generally resistant to HIV infection by blocking virus entry to a cell. Building on this observation, a study published in 2009 reported a potential "cure" in an AIDS patient with leukemia after receiving a bone marrow transplant from a donor with this delta 32 CCR5 mutation. This approach transferred the hematopoietic stem/progenitor cells (HSPC) residing in the bone marrow from the delta 32 donor, and provided a self-renewable and lifelong source of HIV-resistant immune cells. After transplantation, this patient was able to discontinue all anti-HIV drug treatment, the CD4 count increased, and the viral load dropped to undetectable levels, demonstrating an effective transplantation of protection from HIV and suggesting that this approach could have broad clinical utility.

But donors with the delta 32 CCR5 mutation are not generally available, and so how could we engineer an analogous CCR5 negative state in human HSC to be used for bone marrow transplantation, including a patient’s own HSPC? A potential answer comes from zinc finger nucleases (ZFNs) which have been demonstrated to efficiently block the activity of a gene by cleaving the human genome at a predetermined site and altering the genetic sequence via an error-prone DNA repair process. This modification of the cellular DNA is permanent and can fully block gene function. Recently, ZFNs have been shown to inactivate CCR5 in primary human CD4 T cells, allowing them to preferentially survive and expand in the presence of HIV. A human clinical trial evaluating this approach is on-going, in which patient T cells are re-infused after ZFN-treatment to block CCR5 expression and possibly provide an HIV-resistant reservoir of CD4 T cells.

This CIRM Disease Team proposed an approach to modify a patient’s own HSPC to circumvent the need to find matched donors that carry the delta 32 CCR5 mutation and yet provide a renewable and long-lasting source of HIV-resistant cells. Testing of this concept is proposed in selected AIDS lymphoma patients who routinely undergo HSPC transplantation. During the second year of this project, the disease team has made considerable progress and met all the project milestones for year 2. More specifically, the team developed an optimized procedure for efficiently introducing the CCR5-specific ZFNs in HSPC. We showed that these modified cells function normally and retain their “stemness” in tissue culture systems. We also showed these modified cells can be transplanted into mice to reconstitute the immune system. Given HSPC are long lasting stem cells, we have been able to stably detect these cells in mice for over 3 months post-transplantation. The team is in the process of scaling up the cell production procedures to ensure we can generate CCR5-modified HSPC at clinical scale. We are also moving ahead with the remaining pre-clinical safety and efficacy studies required before initiating a clinical trial.

It is well known that infection with HIV-1 requires a protein called CCR5, and persons with a natural mutation in this gene (CCR532) are protected from HIV/AIDS. Everyone has two copies of the CCR5 gene, one inherited from their mother and one from their father. People with both copies of CCR5 mutated (CCR532/ CCR532) are highly resistant to becoming infected with HIV-1. If only one copy is abnormal (CCR5/ CCR532), infection can occur but progression of the infection to AIDS is delayed. The only clear cure of HIV-1 infection occurred in a patient with leukemia who received a blood stem cell transplant from a tissue-matched donor whose cells carried the double mutation CCR532/CCR532. After transplantation, this patient was able to stop all anti-HIV medicine, the immune system improved, and the level of HIV-1 in the blood dropped to undetectable levels. Even after more than 4 years off anti-HIV medicine, the patient is considered cured, as there is no evidence of an active HIV-1 infection.

This Disease Team proposes to treat blood stem cells from an HIV-1 infected person with a protein that can mutate the CCR5 gene, and then transplant these same cells back into the patient to try and reproduce the effects of the CCR532 mutation by providing a renewable and long-lasting source of HIV-1 resistant cells. This will circumvent the need to find a stem cell donor who happens to carry the CCR532/ CCR532 mutation and is a suitable "perfect match" for tissue transplant. The proteins that will be used in this treatment are called Zinc Finger Nucleases (ZFNs). Preliminary results in mice transplanted with ZFN-treated blood stem cells have shown that the modified cells are functional and produce CCR5 mutant progeny cells - including CD4 T cells that are the natural target of HIV-1. Importantly, after HIV-1 infection, the mice demonstrated reduced viral loads, maintenance of CD4 T cells in peripheral tissues, and a powerful survival advantage for the CCR5-negative cells [Holt et al., Nature Biotechnology 2010; 28: 839-47]. These data support the development of this ZFN approach to treat HIV-1 infected patients by first isolating the subjects own blood stem cells, modifying them using CCR5-specific ZFNs, and then re-infusing them back into the patient to thereby reconstitute the immune system with CCR5-mutant, HIV-1 resistant cells. The Disease Team assembled to accomplish this goal has expertise in stem cell technology [City of Hope], HIV-1 infection in pre-clinical mouse models [University of Southern California], and in ZFN-based clinical trial development [Sangamo BioSciences].

In the first two years of study, the Disease Team focused on the use of an existing delivery technology for introducing the ZFNs into blood stem cells. This approach used a type of gene therapy vector called an adenoviral vector, which had been previously used in early stage investigational clinical trials for the modification of patients’ T cells. During this phase of the project, the Disease Team was able to establish a method that allowed the large scale manufacture of ZFN-modified blood stem cells under conditions suitable for a clinical trial. These results were recently published [Li L. et al. Molecular Therapy; advance online publication 16 April 2013]. In year 3 of the study, the Disease Team developed a new method for delivering the ZFNs to the blood stem cells using messenger RNA (mRNA, or SB-728mR). Using a process called electroporation, in a technique that involves exposing a mixture of the blood stem cells and the SB-728mR to a transient electrical field, efficient mutation of the CCR5 gene was achieved. These cells were able to be transplanted into mice, where they engrafted and differentiated to generate human immune cells carrying mutated CCR5 genes. This mRNA-based approach has proven to be robust, well-tolerated and eliminates all viral vector components from the manufacturing process. Thus, electroporation of SB-728mR has now been chosen to move into clinical-scale manufacturing and to support our proposed clinical trial. In Year 4 of the study, the Disease Team will complete the necessary studies to demonstrate the safety of these modified blood stem cells, and submit the required federal and local regulatory documents to support the Phase I clinical trial of this new drug.

Infection with HIV-1 requires entry of the virus into cells by binding to a receptor protein called CCR5. Persons with a natural mutation in the gene encoding CCR5 (CCR532) are protected from HIV-1 infection and AIDS. Everyone has two copies of this gene, one inherited from their mother and one from their father. People with both copies of CCR5 mutated (CCR532/ CCR532) are highly resistant to becoming infected with HIV-1. If only one copy is abnormal (CCR5/ CCR532), infection with HIV can occur but progression of the infection to AIDS is delayed. The only clear cure of HIV-1 infection occurred in a patient with leukemia who received a blood stem cell transplant from a tissue-matched donor whose cells carried the double mutation CCR532/CCR532. After transplantation, this patient was able to stop all anti-HIV medicine, the immune system improved, and the level of HIV-1 in the blood dropped to undetectable levels. Even after more than 6 years off anti-HIV medicine, the patient is considered cured, as there is no evidence of an active HIV-1 infection.

This Disease Team proposed to treat blood stem cells from an HIV-1 infected person with a protein that can mutate the CCR5 gene, and then transplant these same cells back into the patient. The idea was to try and reproduce the effects of the CCR532 mutation by providing a renewable and long-lasting source of these HIV-1 resistant cells. This approach gets around the need to find a stem cell donor who happens to carry the CCR532/ CCR532 double mutation and is a suitable "perfect match" for tissue transplant. The proteins that will be used in this treatment are called Zinc Finger Nucleases (ZFNs). Preliminary results in mice transplanted with ZFN-treated blood stem cells showed that the modified cells are functional and produce CCR5 mutant progeny cells - including CD4 T cells that are the natural target of HIV-1. Importantly, after HIV-1 infection, the mice demonstrated reduced viral loads, maintenance of CD4 T cells in peripheral tissues, and a powerful survival advantage for the CCR5-negative cells [Holt et al., Nature Biotechnology 2010; 28: 839-47]. These data supported the development of this ZFN approach to treat HIV-1 infected patients by first isolating the subjects own blood stem cells, modifying them using CCR5-specific ZFNs, and then re-infusing them back into the patient to thereby reconstitute the immune system with CCR5-mutant, HIV-1 resistant cells. The Disease Team assembled to accomplish this goal has expertise in stem cell technology [City of Hope], HIV-1 infection in pre-clinical mouse models [University of Southern California], and in ZFN-based clinical trial development [Sangamo BioSciences].

The Disease Team developed a new method for delivering the ZFNs to the blood stem cells using messenger RNA (mRNA, or SB-728mR). Using a process called electroporation, a technique that involves exposing a mixture of the blood stem cells and the SB-728mR to a transient electrical field, efficient mutation of the CCR5 gene was achieved. This method was tested in mice and again showed evidence of protection from HIV-1. Electroporation of SB-728mR was then moved into clinical-scale manufacturing to support our proposed clinical trial. In Year 4 of the study, the Disease Team completed studies demonstrating the safety of these modified blood stem cells, and obtained the required federal and local approvals to initiate the first-in-human testing of the CCR5-modified blood stem cell therapy. The Phase I clinical trial, which is sponsored by City of Hope and funded by Sangamo BioSciences and CIRM under its Strategic Partnership funding mechanism, is planned to start accrual in the fourth quarter of 2014.